| Part ⅡTNF-a and fg12expression in rat myocardial ischemia/reperfusion injuryObjective:Proinflammatory cytokine tumor necrosis factor-alpha(TNF-a) during myocardial ischemia/reperfusion(MI/R) injury has been studied extensively. However, effect TNF-a in microvascular dysfunction of MI/R is still unclear. This study investigates TNF-a and fgl2expression in rat myocardial ischemia/reperfusion(MI/R), and elucidates the relationship between TNF-a and fgl2in MI/R, which explore the mechanism of TNF-a-induced myocardial ischemia/reperfusion.Methods:Myocardial ischemia/reperfusion model was established for this study. Sprague-Dawley rats were randomly divided into four groups:sham group, one-day group, three-day group, seven-day group with MI/R(Twelve rats per group). After30minutes of ligation of the left anterior descending coronary artery, the hearts were reperfused for one, three and seven days. Twelve rats were subjected to the same process without performing occlusions as sham.. Another36rats were administered either with nonimmune IgG or the neutralizing antibodies to TNF-a (three hours prior to initiating I/R)(each group consists of Twelve rats). Myocardial infarct size was assessed with Evans blue and TTC staining. Western blot was performed for TNF-a protein expression, fg12protein expression and fibrin accumulation.Results:Evans blue and TTC staining showed that the infarct size in three-day group in the MI/R group was significantly larger than that in three-day group and seven-day group. TNF-a and fg12protein expression were increased after MI/R. TNF-a and fgl2protein expression in three-day group were significantly higher compared with one-day group and seven-day group. fg12expression was downregulated accompanied with antibody neutralization of TNF-a.Conclusion:TNF-a and fg12protein expression are upregulated in MI/R. Upregulation of fg12expression is related with increasing of TNF-a expression in MI/R. Part ⅡEffect of TNF-a on fg12in cardiac microvascular endothelial cellsObjective:The pathophysiology of MI/R injury is complicated. Tumor necrosis factor-a (TNF-a) as proinflammatory cytokine plays a crucial role in MI/R injury. Endothelial dysfunction may be one of the mechanisms that contribute to microvascular dysfunction during MI/R injury. The present experiments explore that effect of TNF-a on fg12in cardiac microvascular endothelial cells, and the role of TNF-a-induced fgl2expression in mechanism of microthrombosis during MI/R injury. Methods: Microvascular endothelial cells were isolated from neonatal rat heat. CMECs were stimulated with various concentrations of TNF-α(12.5ng/ml,25ng/ml,50ng/ml) for24h, and exposed to TNF-α(25ng/ml) for12h,24h,48h. Fgl2mRNA expression was measured by Real-time quantitative PCR. Fgl2protein expression was assessed by Western blot. Confocal laser scanning microscopy(CLSM) was used for fgl2immunofluorescent staining. Thrombin generation assays was carried out to test the ability of CMECs to generate thrombin.Results:fgl2mRNA and protein were increased after CMECs were treated with TNF-α. Accordingly, the ability to generate thrombin was upregualted in CMECs after TNF-α.treatment. CLSM showed that fgl2protein was abundantly expressed on the membrane.Conclusion:TNF-α can induce fgl2expression in CMECs, which can be one of mechanisms that contribute to microthrombosis of microvascular dysfunction in rat MI/R injury. Part ⅢEvalutation of signal transduction pathways for TNF-a-induced fgl2in cardiac microvascular endothelial cellsObjective:Numerous studies have shown that NF-κB and p38MAPK signal transduction pathway are involved in MI/R injury. However, the effect of NF-κB and p38MAPK in microvascular dysfunction during MI/R injury is still unclear. This experiment explores the mechamism of signal transduction pathways for TNF-a-induced fg12in cardiac microvascular endothelial cells.Methods:Microvascular endothelial cells were isolated from neonatal rat heat. PDTC, one specific and widely-used NF-κB inhibitor, SB203580, one p38MAPK-specific inhibitor were used to pretreat CMECs for one hour, then CMECs were stimulated with TNF-a(25ng/ml) for24hours. Fg12protein expression was measured by Western blot.Results:After CMECs with PDTC prteatment, fg12protein expression was decreased compared to CMECs without PDTC prteatment. Similarily, fgl2protein expression was reduced when CMECs were preteated with SB203580(10μmol/1) for one hour. TNF-a-induced fg12expression was significantly downregulated by a combination of PDTC and SB203580compared to when they were used alone.Conclusion:TNF-a can induce fgl2expression via activiation of NF-κB and p38MAPK signal transduction pathway in CMECs, which results in the formation of fibrin-rich microthrombus in rat MI/R injury. |
PDF Full Text Request